Plant Disease

In the fall of 2015 and 2016, during annual disease surveys conducted by the Pennsylvania Department of Agriculture, seven peach trees (Prunus persica) from a commercial orchard in Berks County, PA, were observed exhibiting symptoms consistent with phytoplasma infection. Symptoms included premature leaf yellowing and reddening, foliar epinasty, shotholes and vein reddening. Seven out of 700 peach trees in the orchard displayed these symptoms and were ultimately removed. Leaf and stem samples were collected from seven symptomatic trees as well as from five asymptomatic ones for further analysis. Total DNA was extracted using the Qiagen DNeasy Plant Kit. All DNA samples were initially screened using a phytoplasma-specific real-time PCR assay (Hodgetts et al., 2009). Phytoplasma was detected in all symptomatic plants, but in none of the five asymptomatic plants. Positive samples were subsequently used in conventional nested PCR targeting the 16S rRNA gene, using primer pairs P1/16S-SR followed by P1A/16S-SR (Deng & Hiruki, 1991; Lee et al., 2004). PCR products were cloned, and sequenced. Four representative sequences were deposited in GenBank under accession numbers PV770936 to PV770939. Sequence analysis using BLASTn (NCBI) and iPhyClassifier (Zhao et al., 2009) revealed that the phytoplasma strains, designated as PYP-PA03, 04, 06, and 07, shared between 97.5% (1,482/1,520 bp) and 97.63% (1,483/1519 bp) sequence identity with the ‘Candidatus Phytoplasma fraxini’ reference strain 16SrVII-A (AF092209), suggesting that the peach associated phytoplasma is a ‘Ca. P. fraxini’-related strain. However, virtual RFLP analysis of the 16S rDNA F2nR2 fragments revealed patterns distinct from all previously established 16Sr groups/subgroups. The closest similarity was observed with members of the 16Sr group VI, but similarity coefficients were below the accepted subgroup threshold (0.97), supporting the distinctiveness of these strains. To further characterize the PYP-PA phytoplasma, partial sequences of the tuf and secA genes were PCR-amplified, cloned, and sequenced following protocols by Makarova et al. (2012) and Dickinson & Hodgetts (2013). The resulting sequences were identical for each gene, and one sequence was deposited in GenBank under accession numbers PV769898 (tuf gene) and PV769899 (secA gene). BLASTn analysis of the tuf gene revealed 93.25% (470/504 bp) and 90.67% (457/504 bp) sequence identity with the homologous gene in the Argentinian alfalfa witches’-broom phytoplasma isolate ArAWB-2021 16SrVII-C (CP131022) and the ‘Ca. P. fraxini’ isolate AshY1 16SrVII-A (CP146843), respectively. Similarly, the secA gene shared 89.16% (691/775 bp) and 88.30% (679/769 bp) sequence identity with the same isolates ArAWB-2021 (CP131022) and AshY1 (CP146843), respectively. These collective results suggest that the PYP-PA phytoplasma is phylogenetically related to members of the Ash yellows group (16SrVII). In 2010, Zunnoon‐Khan et al. identified a phytoplasma strain PRU0430 of group 16SrVII associated with peach trees in Ontario, Canada (GU223903). To our knowledge, the present study is the first to report a ‘Ca. P. fraxini’-related strain associated with peach trees in the United States. These findings highlight the need for further research to fully characterize this potentially novel phytoplasma, which constitutes a distinct lineage from the one identified in Canada and underscore the importance of continued surveillance and molecular investigation of emerging phytoplasma-associated diseases in the region.

Buckwheat (Fagopyrum esculentum Moench) is a fast-growing, low-input crop in the Polygonaceae family, cultivated on about 2 million hectares worldwide, mainly in Russia, China, and Ukraine by FAO (2023). In Taiwan, it is grown as a winter cover crop during fallow periods and is valued for improving soil health, suppressing weeds, and supporting sustainable farming systems. It is also utilized in various food products due to its rich fiber content and gluten-free properties. In May 2024, 147 out of 3000 plants (Taichung No. 5) corresponding to an incidence rate of approximately 5% exhibiting floral abnormalities, including phyllody, virescence, and flower sterility, were observed in Xinshe District in Taichung, central Taiwan (Fig. 1). These symptoms were indicative of possible phytoplasma infection. For further investigation, floral and leaf tissues were randomly collected from 10 symptomatic and 10 asymptomatic buckwheat plants. Plant total DNA was extracted using the DNeasy Plant Mini Kit, and PCR was performed targeting the 16S rRNA gene using universal phytoplasma primers P1A/P7A, followed by a semi-nested reaction with P1A/16S-SR (Lee et al. 2004). All PCR amplicons of approximately 1.5 kb were obtained only from symptomatic samples. PCR amplicons were sequenced by Sanger sequencing, and sequences were assembled by BioEdit. All symptomatic samples yielded identical sequences and a representative sequence (1,543 bp, nearly full-length 16S rRNA gene) was deposited in GenBank (Accession no. PV487329). Based on virtual RFLP analysis using iPhyClassifier (Zhao et al. 2009), the phytoplasma strain detected in diseased buckwheat plants was identified as a ‘Candidatus Phytoplasma aurantifolia/citri’-related strain, sharing 98.27% sequence identity with the reference strain (witches broom disease of lime, WBDL, Accession no. U15442). The strain, designated as Buckwheat Phyllody (BWP) TSIPS 3-1, was classified within the 16SrII-A subgroup, with a similarity coefficient of 1.00 when compared to the reference strain of subgroup 16SrII-A (Accession no. L33765). A BLASTn search against the NCBI database revealed that BWP-TSIPS 3-1 shared 99.94% identity with the 16S rRNA gene of potato purple top phytoplasma (Accession no. KM212951, a ‘Ca. P. aurantifolia/citri’-related strain), identified in Guangdong, China (Cheng et al. 2019). To further confirm phytoplasma identity, an additional gene marker, secY gene was amplified by semi-nested PCR using group-specific primers SecYF1(II)/SecYR1(II) and SecYF2(II) /SecYR1(II) (Lee et al. 2010). The 1,263-bp amplicons were obtained from all symptomatic samples. Sequencing results revealed identical secY sequences across samples, and a representative sequence was deposited in GenBank (Accession no. PV567544). BLASTn analysis indicated 100% (1263/1263) identity with the secY gene of the phytoplasmas, including black gram witches’-broom phytoplasma in Myanmar (Accession no. AB703249), which is classified within the 16SrII-A subgroup (Win and Jung, 2012). Phytoplasma infections associated with buckwheat have been rarely reported, with only a single case documented in India, though the short 16S rRNA gene sequence limited subgroup classification (Bandakkanavar et al. 2023). Similar symptoms, such as phyllody, have also been observed in Ukraine, but the causal agent remains unconfirmed due to the absence of molecular diagnostics (Demchenko et al. 2016). Our study confirmed the first identification of a ‘Ca. P. aurantifolia/citri’-related strain (16SrII-A subgroup) in buckwheat in Taiwan. Phytoplasmas of the 16SrII-A subgroup have been widely documented across Taiwan, including in Taichung (Tseng et al. 2012). Given the rising popularity of buckwheat for its dietary benefits and its increasing incorporation into crop rotation systems, especially as a winter cover crop, the presence of phytoplasma presents potential concerns. Infected buckwheat plants may inadvertently serve as hosts or reservoirs for phytoplasmas and their insect vectors, indicating a need for ongoing monitoring, especially in areas where buckwheat is part of crop rotations.

Areca palm yellow leaf phytoplasma (AYLP) and Areca palm velarivirus 1 (APV1) are two critical pathogens associated with yellow leaf in areca palm, resulting in devastating damage to areca production. However, evidence of their co-infection remains unclear. Areca palms showing yellow leaf symptoms (suspected to be caused by phytoplasma and velarivirus 1) were surveyed and sampled around Hainan Island of China and the pathogens were identified and analyzed in the study. The results showed that infectious leaf yellowing symptoms of areca palms primarily occurred in eastern and southern cities and counties of Hainan Island. Molecular identification revealed co-infection of 'Candidatus Phytoplasma asteris' (16SrI-B subgroup) and APV1 in individual areca palm samples. Among 520 samples tested by qPCR, AYLP was detected in 23.46% (n=122), APV1 in 53.46% (n=278), and a co-infection with both AYLP and APV1 in 10.96% (n=57). A statistically significant difference in APV1 detection rate was observed between asymptomatic and symptomatic plantations (P < 0.0001), suggesting APV1 infection likely represents an important risk factor for yellowing development in cultivated areca palm populations. These results confirm the co-infection of AYLP and APV1 on Hainan Island, offering epidemiological evidence for targeted disease management.

Lethal wilt (LW), also known as “Marchitez Letal (ML)” in Colombia, is an endemic disease affecting oil palms (Elaeis guineensis Jacq.) and is a leading cause of crop loss. The disease is characterized by the drying of leaflets from the tip to the base, primarily impacting the lower third of the plant and progressively moving upward. This progression leads to physiological disturbances, including necrosis at the tips of immature inflorescence bracts (spines) and the detachment of bunch fruits, ultimately causing wilting. As a phytosanitary measure, infected palms are eradicated to prevent further spread of the disease. The primary goal of this research was to identify the bacteria associated with LW and to validate a molecular detection method. A 16S amplicon-based analysis was employed to identify and compare the microbial diversity in LW-affected palm tissues with those of healthy plants. Among the 16 OTUs corresponding to different bacterial genera found in all LW samples, taxonomic classification and symptomatology suggested that the bacteria closely associated with LW belong to the genus Candidatus Liberibacter. Further phylogenetic analysis indicated that these bacteria are part of the Rhizobiaceae family, grouping closely with other species of the genus Candidatus. Liberibacter. The concentration of the pathogen in different oil palm tissues was determined using droplet digital PCR (ddPCR) and quantitative PCR (qPCR), expressed in copies/µL in the LW samples. This study represents the first report of ‘Candidatus Liberibacter sp’. being associated with lethal wilt in oil palms of the Arecaceae family in Colombia. The findings from this research have the potential to contribute significantly to the development of effective management strategies to prevent crop losses.

The Asian citrus psyllid (ACP) is the vector of ‘Candidatus Liberibacter asiaticus’ (CLas), the causal agent of citrus greening or Huanglongbing (HLB), one of the most devastating citrus diseases worldwide. The citrus industry in Georgia (U.S.A.) is in the process of a rapid expansion, and based on experiences with HLB in Florida, there is great concern about the potential impacts of HLB on this emerging industry. Prior to 2023, ACP had been identified in residential citrus trees in isolated Georgia counties, but little to no testing of psyllids for CLas had occurred. However, in 2023, one individual psyllid collected from Chatham County was confirmed positive for CLas by PCR and sequencing. Furthermore, during 2023, ACP adults and nymphs were identified for the first time in a Georgia commercial citrus grove. The finding of ACP in a commercial planting represents a significant risk for CLas dissemination and thereby has the potential to stall the rapid expansion of Georgia’s citrus industry. In the coming years, surveillance and testing of ACP from commercial groves will be essential for the early detection and management of HLB and its vector to reduce HLB spread within Georgia’s commercial groves.

The occurrence of ‘Candidatus Liberibacter’ spp. and ‘Ca. Phytoplasma’ spp. associated with blotchy mottle symptoms poses challenges to huanglongbing (HLB) diagnosis using molecular techniques. The ability to detect multiple targets simultaneously and specifically is a key aspect met by quantitative PCR (qPCR). A set of primers and hydrolysis probes useful in either single or multiplex reactions for the detection and quantification of HLB-associated bacteria were developed. Sequences from conserved genes of the ribosomal proteins for Liberibacter and phytoplasma circumvent the lack of specificity and cross-reactivity problems related to 16Sr DNA gene amplification, allowing precise and specific detection of HLB-associated bacteria in citrus and in the Liberibacter vector, Diaphorina citri. The triplex reaction exhibited high quality and precision as a robust tool for quantifying ‘Ca. L. asiaticus’ (CLas), ‘Ca. L. americanus’ (CLam), and 16Sr IX phytoplasma. Triplex qPCR showed consistent results and comparable sensitivity to the ribonuclease reductase test, although quantification cycle (Cq) values were higher when compared with 16SrDNA qPCR. Detection tests using field samples indicate that the qPCR triplex can identify HLB-associated bacteria in samples with varying levels of symptoms, ranging from typical to asymptomatic. Assessment of field samples from growers indicated more than 78.6% had Cq lower than 35.0, below the cutoff established for qPCR reactions used in this work. qPCR triplex is a safe, specific, and sufficiently sensitive technique for detecting CLas, CLam, and 16Sr IX phytoplasma simultaneously, in both citrus and D. citri samples. Its application is of importance in assisting growers in making decisions for HLB management.